Antibiotic resistance and evasion of treatment are increasing at alarming rates for numerous major bacterial pathogens, designated as ?superbugs,? including ovoid-shaped (ovococcus) pathogens, such as Streptococcus pneumoniae (pneumococcus). The regulation of peptidoglycan (PG) homeostasis and PG cell wall stress responses contains potential vulnerabilities that can be exploited to discover new antibiotics. Considerable progress has recently been made in determining the mechanisms, components, enzymology, and dynamics of PG synthesis and its coordination with cell division in a wide variety of bacteria, including S. pneumoniae. However, relatively little is specifically known about how ovococcus Gram-positive pathogens regulate PG homeostasis and respond to PG cell wall stresses, especially in comparison to non-pathogenic model bacteria. A major goal of this proposal is to fill in this large knowledge gap. The premise of this proposal is that there are uncharacterized, interlinked regulatory circuits that mediate pneumococcal PG homeostasis and cell wall stress responses and are required for colonization and virulence of its human host. This premise is based on the new discovery of an interconnected regulatory network that links PG synthesis, cell division, second messenger signaling, and responses to PG cell wall stress in S. pneumoniae. This network was serendipitously found by unbiased genetic selections for suppressors of mutations in essential genes required for PG synthesis. Notably, the regulatory proteins identified in this network are conserved in other pathogens and were previously identified as uncharacterized colonization and virulence factors of S. pneumoniae. The long-term goal of this project is to use the ?superbug? Streptococcus pneumoniae as a highly tractable ovococcus experimental model to understand the mechanisms, functions, and interconnections of this important new regulatory network of PG homeostasis. The network consists of three interconnected parts that are the subjects of the three specific aims of this proposal. Aim I. Determine the functions of a newly discovered, multi-subunit RNA-binding protein, that likely acts as a post-transcriptional regulator, in setting cell size, inducing cell wall stress responses, and in biofilm formation. Aim II. Determine the functions of the second messenger, c-di-AMP, in suppressing essential PG synthesis mutations, in regulating PG homeostasis and stress responses, and in capsule and biofilm formation. Aim III. Determine mechanisms that link regulation by the RNA-binding protein and the c-di-AMP second messenger to WalRK two-component system (TCS) sensing and responses to cell wall stress. Combined results and conclusions from this proposal will lead to new paradigms and models for the regulation of PG cell wall homeostasis and stress responses relevant to the pathogenesis of S. pneumoniae, and likely other ovococcus pathogens. In addition, this proposal will produce important information about the mechanisms, functions, and interconnections of a new regulatory network that has the potential to provide targets for future antibiotic discovery.